CCR3 inhibition for ocular angiogenesis and macular degeneration

ABSTRACT

Provided are methods and compositions for the treatment or prevention of ocular angiogenesis and neovascularization. Administration of inhibitors of the CCR3 receptor or its ligands eotaxin (CCL11), eotaxin-2 (CCL24) or eotaxin-3 (CCL26) inhibits ocular angiogenesis.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. application Ser. No.11/357,288, filed Feb. 16, 2006, the disclosures of which are expresslyincorporated by reference herein.

FIELD OF THE INVENTION

The present invention relates to the suppression of ocular angiogenesisby inhibiting the CCR3 receptor.

DESCRIPTION OF THE RELATED ART

The macula is the part of the retina which is responsible for centralvision. Age-related macular degeneration is a chronic eye disease thatoccurs when tissue in the macula deteriorates. Macular affects centralvision, but not peripheral vision. Macular degeneration is the leadingcause of severe vision loss in people age 60 and older.

There are two forms of age-related macular degeneration: dry and wet.Dry macular degeneration is the most common type of macular degenerationand occurs when cells of the macula slowly begin to break down. Yellowdeposits called “drusen” form under the retina between the retinalpigmented epithelium (RPE) and Bruch's membrane, which supports theretina. The drusen deposits are debris associated with compromised cellmetabolism in the RPE. Eventually there is a deterioration of themacular regions associated with the drusen deposits resulting in a lossof central vision.

Wet macular degeneration occurs when abnormal bold vessels grow behindthe macula. These vessels are fragile and can leak fluid and blood,which result in scarring of the macula and raise the potential forrapid, severe damage. Bruch's membrane breaks down, usually near drusendeposits. This is where new blood vessel growth, or neovascularization,occurs. Central vision can become distorted or lost entirely in a shortperiod of time, sometimes within days. Wet macular degeneration isresponsible for about 10 percent of the cases of age-related maculardegeneration, but it accounts for about 90 percent of the cases of legalblindness.

SUMMARY OF THE INVENTION

The present invention relates to a method of inhibiting ocularangiogenesis. The method comprises exposing a choroidal cell to aCCR3-inhibitory effective amount of a compound which inhibits theactivity of CCR3. The present invention also relates to a compositionfor the inhibition of ocular angiogenesis. The composition comprises acompound which inhibits the activity of CCR3.

Other systems, methods, features and advantages of the present inventionwill be or become apparent to one with skill in the art upon examinationof the following drawings and detailed description. It is intended thatall such additional systems, methods, features and advantages beincluded within this description, be within the scope of the presentinvention, and be protected by the accompanying claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the effect of laser injury on the number of CCR3receptors on choroidal endothelial cells.

FIG. 2 shows the effect of CCR3 antibody on the proliferation ofchoroidal endothelial cells following laser injury.

FIG. 3 illustrates the dose-dependent effect of CCR3 antibody onchoroidal neovascularization volume

FIG. 4 shows lack of infiltration of eosinophils and mast cells into thechoroid following laser injury.

FIG. 5 illustrates the lack of change of number of infiltratingmacrophages in the choroid following laser injury and CCR3 antibodytreatment.

DESCRIPTION OF PREFERRED EMBODIMENTS

Intraocular inflammation is not clinically apparent in age-relatedmacular degeneration. However, there is evidence suggesting aninfluential role for inflammation in this condition. CCR3 is apromiscuous chemokine receptor that is predominantly expressed byeosinophils but also is found on other leukocytes and some endothelialand epithelial cells.

The invention relates to methods and compositions for the treatment orprevention of ocular angiogenesis and neovascularization. Administrationof inhibitors of the CCR3 receptor or its ligands, for example eotaxin(CCL11), eotaxin-2 (CCL24) or eotaxin-3 (CCL26), inhibits ocularangiogenesis. Ocular angiogenesis includes choroidal angiogenesis andretinal angiogenesis. Compositions and methods for inhibiting CCR3,eotaxin (CCL11), eotaxin-2 (CCL24), and eotaxin-3 (CCL26) for thetreatment and/or prevention of neovascular disease are provided. Alsoprovided are novel therapeutic targets and diagnostic markers forchoroidal neovascularization.

Any compound which inhibits the activity of CCR3 may be used in thepresent invention. Such compounds include inhibitory molecules whichbind directly to the CCR3 receptor, antibodies which bind the CCR3receptor or to the natural ligands of the CCR3 receptor, includingeotaxin (CCL11), eotaxin-2 (CCL24) and eotaxin-3 (CCL26), RNA, DNA orRNA/DNA aptamers which specifically bind CCR3, eotaxin, eotaxin-2 oreotaxin-3, and siRNA or anti-sense oligonucleotides which inhibit theexpression of CCR3, eotaxin, eotaxin-2 or eotaxin-3.

Numerous “small molecule” inhibitors for the CCR3 receptor have beendeveloped and can be used in the present invention. In one aspect theCCR3 inhibitor is an organic molecule having a molecular weight lessthan 1000. In another aspect of the invention, the CCR3 inhibitor is anorganic molecule having a molecular weight less than 500. The CCR3receptor inhibitors include piperidine derivatives, piperidine amidesand biperidine compounds such as those described in U.S. Pat. Nos.6,984,651 and 6,903,115, and U.S. published applications 20050176708,20050182094 and 20050182095; heterocyclic piperidines such as thosedescribed in U.S. Pat. No. 6,759,411; diphenyl-piperidine derivativessuch as those described in U.S. Pat. No. 6,566,376; 2,5-substitutedpyrimidine derivatives such as those described in U.S. Pat. No.6,984,643; piperizinones such as those described in U.S. Pat. No.6,974,869; bicycylic and tricyclic amines such as those described inU.S. Pat. No. 6,960,666; N-ureidoalkyl-piperidines such as thosedescribed in U.S. Pat. Nos. 6,949,546, 6,919,368, 6,906,066, 6,897,234,6,875,776, 6,780,857, 6,627,629, 6,521,592 and 6,331,541; bicyclicdiamines such as those described in U.S. Pat. No. 6,821,964;benzylcycloalkyl amines such as those described in U.S. Pat. No.6,864,380; 2-substituted-4-nitrogen heterocycles such as those describedin U.S. Pat. No. 6,706,735; ureido derivatives ofpoly-4-amino-2-carboxy-1-methyl pyrrole compounds; bicyclic and bridgednitrogen heterocycles such as those described in U.S. publishedapplication 20050234034; azetidine derivatives such as those describedin U.S. published application 20050222118; substituted fused bicyclicamines such as those described in U.S. published application20050197373; substituted spiro azabicyclics such as those described inU.S. published application 20050197325; piperidine-substituted indolesor heteroderivatives thereof such as those described in U.S. publishedapplication 20050153979; piperidinyl and piperazinyl compoundssubstituted with bicyclo-heterocyclylalkyl groups such as thosedescribed in U.S. published application 20050090504; arylsulfonamidederivatives such as those described in U.S. published application20050070582; 1-phenyl-1,2-diaminoethane derivatives such as thosedescribed in U.S. published application 20040063779;(N-{[2S]-4-(3,4-dichlorobenzyl)morpholin-2-yl}methyl)-N′[(2-methyl-2H-tetraazol-5-yl)methyl]urea) (see, e.g., Nakamura et al.,Immunol Res., 33:213-222, 2006;N-{(3R)-1-[(6-fluoro-2-naphthyl)methyl]pyrrolidin-3-yl}-2-{1-[(3-methyl-1-oxidopyridin-2-yl)carbonyl]piperidin-4-ylidene}acetamide(see, e.g., Suzuki et al., Biochem. Biophys. Res. Commun.,339:1217-1223, 2006;N-{(3R)-1-[(6-fluoro-2-naphthyl)methyl]pyrrolidin-3-yl}-2-{1-[(5-hydroxy-3-methylpyridin-2-yl)carbonyl]piperidin-4-ylidene}acetamidehemifumarate (see, e.g., Morokata et al., J. Pharmacol. Exp. Ther., Dec.9, 2005 [Epub ahead of print]); bipiperidine amide antagonists of CCR3such as those described in Ting et al., Bioorg. Med. Chem. Lett.,15:3020-3023, 2005;(S)-methyl-2-naphthoylamino-3-(4-nitrophenyl)propionate (see, e.g.,Beasley et al., J. Allergy Clin. Immunol., 105: S466-S472, 2000; and theCCR3 antagonist compounds described in Fryer et al., J. Clin. Invest.,116:228-236, 2006.

Additional compounds for inhibiting the CCR3 receptor include RNA, DNAor RNA/DNA aptamers directed against CCR3, eotaxin, eotaxin-2 oreotaxin-3. Exemplary methods for making aptamers are described in U.S.Pat. Nos. 5,270,163, 5,840,867, 6,180,348 and 6,699,843.

Additional compounds for inhibiting the CCR3 receptor include anti-senseoligonucleotides or siRNAs directed against CCR3, eotaxin, eotaxin-2 oreotaxin-3, including the anti-sense oligonucleotides directed againstthe CCR3 receptor such as that described in U.S. Pat. No. 6,822,087.

The siRNAs for use in the present invention are designed according tostandard methods in the field of RNA interference. Introduction ofsiRNAs into cells may be by transfection with expression vectors, bytransfection with synthetic dsRNA, or by any other appropriate method.Transfection with expression vectors is preferred.

The expression vectors which can be used to deliver siRNA according tothe invention include retroviral, adenoviral and lentiviral vectors. Theexpression vector includes a sequence which codes for a portion of thetarget gene (e.g., CCR3 receptor, eotaxin, eotaxin-2 or eotaxin-3) whichis to be silenced. The target gene sequence is designed such that, upontranscription in the transfected host, the target RNA sequence forms ahairpin structure due to the presence of self-complementary bases.Processing within the cell removes the loop resulting in formation of asiRNA duplex. The double stranded RNA sequence should be less than 30nucleotide bases; preferably the dsRNA sequence is 19-25 bases inlength; more preferably the dsRNA sequence is 20 nucleotides in length.

The expression vectors may include one or more promoter regions toenhance synthesis of the target gene sequence. Promoters which can beused include CMV promoter, SV40 promoter, promoter of mouse U6 gene, andpromoter of human H1 gene.

One or more selection markers may be included to facilitate transfectionwith the expression vector. The selection marker may be included withinthe expression vector, or may be introduced on a separate geneticelement. For example, the bacterial hygromycin B phosphotransferase genemay be used as a selection marker, with cells being grown in thepresence of hygromycin to select for those cells transfected with theaforementioned gene.

Synthetic dsRNA may also be introduced into cells to provide genesilencing by siRNA. The synthetic dsRNAs are less than 30 base pairs inlength. Preferably the synthetic dsRNAs are 19-25 base pairs in length.More preferably the dsRNAs are 19, 20 or 21 base pairs in length,optionally with 2-nucleotide 3′ overhangs. The 3′ overhangs arepreferably TT residues.

Synthetic dsRNAs can be introduced into cells by injection, bycomplexing with agents such as cationic lipids, by use of a gene gun, orby any other appropriate method.

Additional compounds for inhibiting the CCR3 receptor include antibodieswhich specifically bind the CCR3 receptor, eotaxin, eotaxin-2 oreotaxin-3. Exemplary antibodies which specifically bind and inhibit theCCR3 receptor are described in U.S. Pat. Nos. 6,806,061 and 6,207,155,and in U.S. published applications 20050191702, 20050069955, and20020147312. Exemplary antibodies which specifically bind and inhibiteotaxin and eotaxin-2 are described in U.S. Pat. Nos. 6,946,546 and6,635,251, and in U.S. published applications 20040191255 and20040014132.

The antibodies of the present invention can be polyclonal or monoclonal,and the term antibody is intended to encompass both polyclonal andmonoclonal antibodies. Antibodies of the present invention can be raisedagainst an appropriate immunogen, including proteins or polypeptides ofthe present invention, such as isolated and/or recombinant mammalianCCR3 receptor, eotaxin, eotaxin-2 or eotaxin-3 protein or portionthereof, or synthetic molecules, such as synthetic peptides.

Preparation of immunizing antigen, and polyclonal and monoclonalantibody production can be performed using any suitable technique. Avariety of methods have been described (see e.g., Kohler et al., Nature,256: 495-497 (1975) and Eur. J. Immunol. 6: 511-519 (1976); Milstein etal., Nature 266: 550-552 (1977); Koprowski et al., U.S. Pat. No.4,172,124; Harlow, E. and D. Lane, 1988, Antibodies: A LaboratoryManual, (Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y.);Current Protocols In Molecular Biology, Vol. 2 (Supplement 27, Summer'94), Ausubel, F. M. et al., Eds., (John Wiley & Sons: New York, N.Y.),Chapter 11, (1991)). Generally, a hybridoma is produced by fusing asuitable immortal cell line (e.g., a myeloma cell line such as SP2/0)with antibody producing cells. The antibody producing cell, preferablythose of the spleen or lymph nodes, are obtained from animals immunizedwith the antigen of interest. The fused cells (hybridomas) are isolatedusing selective culture conditions, and cloned by limiting dilution.Cells which produce antibodies with the desired specificity are selectedby a suitable assay (e.g., ELISA).

Single chain antibodies, and chimeric, humanized or primatized(CDR-grafted) antibodies, as well as chimeric or CDR-grafted singlechain antibodies, comprising portions derived from different species,are also encompassed by the present invention and the term “antibody”.The various portions of these antibodies can be joined togetherchemically by conventional techniques, or can be prepared as acontiguous protein using genetic engineering techniques. For example,nucleic acids encoding a chimeric or humanized chain can be expressed toproduce a contiguous protein. See, e.g., Cabilly et al., U.S. Pat. No.4,816,567; Cabilly et al., European Patent No. 0,125,023. B1; Boss etal., U.S. Pat. No. 4,816,397; Boss et al., European Patent No. 0,120,694B1; Neuberger, M. S. et al., WO 86/01533; Neuberger, M. S. et al.,European Patent No. 0,194,276 B1; Winter, U.S. Pat. No. 5,225,539; andWinter, European Patent No. 0,239,400 B1. See also, Newman, R. et al.,BioTechnology, 10: 1455-1460 (1992), regarding primatized antibody, andLadner et al., U.S. Pat. No. 4,946,778 and Bird, R. E. et al., Science,242: 423-426 (1988)) regarding single chain antibodies.

In addition, functional fragments of antibodies, including fragments ofchimeric, humanized, primatized or single chain antibodies, can also beproduced. Functional fragments of foregoing antibodies retain at leastone binding function and/or modulation function of the full-lengthantibody from which they are derived. For example, antibody fragmentscapable of binding to a mammalian CCR3 receptor, eotaxin, eotaxin-2 oreotaxin-3 or portion thereof, including, but not limited to, Fv, Fab,Fab′ and F(ab′).sub.2 fragments are encompassed by the invention. Suchfragments can be produced by enzymatic cleavage or by recombinanttechniques. For instance, papain or pepsin cleavage can generate Fab orF(ab′).sub.2 fragments, respectively. Alternatively, antibodies can beproduced in a variety of truncated forms using antibody genes in whichone or more stop codons has been introduced upstream of the natural stopsite. For example, a chimeric gene encoding a F(ab′).sub.2 heavy chainportion can be designed to include DNA sequences encoding the CH.sub.1domain and hinge region of the heavy chain.

The antibodies of the present invention can be used to modulate receptoror ligand function in research and therapeutic applications. Forinstance, antibodies can act as inhibitors to inhibit (reduce orprevent) (a) binding (e.g., of a ligand, a second inhibitor or apromoter) to the receptor, (b) a receptor signalling, (c) and/or astimulatory function. Antibodies which act as inhibitors of receptorfunction can block ligand or promoter binding directly or indirectly(e.g., by causing a conformational change). For example, antibodies caninhibit receptor function by inhibiting binding of a ligand, or bydesensitization (with or without inhibition of binding of a ligand).

Anti-idiotypic antibodies are also provided. Anti-idiotypic antibodiesrecognize antigenic determinants associated with the antigen-bindingsite of another antibody. Anti-idiotypic antibodies can be preparedagainst a second antibody by immunizing an animal of the same species,and preferably of the same strain, as the animal used to produce thesecond antibody. See e.g., U.S. Pat. No. 4,699,880. Single chain, andchimeric, humanized or primatized (CDR-grafted), as well as chimeric orCDR-grafted single chain anti-idiotypic antibodies can be prepared, andare encompassed by the term anti-idiotypic antibody. Antibody fragmentsof such antibodies can also be prepared.

Modulation of mammalian CCR3 receptor function according to the presentinvention, through the inhibition of at least one functioncharacteristic of a mammalian CCR3 receptor, provides an effective andselective way of inhibiting ocular angiogenesis. One or more inhibitorsof CCR3 receptor function, such as those identified as described herein,can be used to inhibit ocular angiogenesis for therapeutic purposes.

Thus, the present invention provides a method of inhibiting ocularangiogenesis in an individual in need of such therapy, comprisingadministering a compound which inhibits mammalian CCR3 receptor functionto an individual in need of such therapy. Such individuals include thosehaving age-related macular degeneration.

The methods of the present invention can be used in any mammalianspecies, including human, monkey, cow, sheep, pig, goat, horse, mouse,rat, dog, cat, rabbit, guinea pig, hamster and horse. Humans arepreferred.

According to the method of the invention, one or more compounds can beadministered to the host by an appropriate route, either alone or incombination with another drug. An effective amount of a compound (e.g.,a small molecule CCR3 receptor antagonist which inhibits ligand binding,an antibody or an siRNA) is administered. An effective amount is anamount sufficient to achieve the desired therapeutic effect, under theconditions of administration, such as an amount sufficient forinhibition of a CCR3 receptor function, and thereby inhibition of ocularangiogenesis.

A variety of routes of administration are possible including, but notnecessarily limited to oral, dietary, topical, parenteral (e.g.,intravenous, intraarterial, intramuscular, subcutaneous injection),inhalation (e.g., intrabronchial, intranasal or oral inhalation,intranasal drops), and intraocular injection routes of administration,depending on the disease or condition to be treated. Intraocularinjection routes include periocular (subconjunctival/transscleral),intravitreous, subretinal and intracameral modes of injection.

Formulation of a compound to be administered will vary according to theroute of administration selected (e.g., solution, emulsion, capsule). Anappropriate composition comprising the compound to be administered canbe prepared in a physiologically acceptable vehicle or carrier. Forsolutions or emulsions, suitable carriers include, for example, aqueousor alcoholic/aqueous solutions, emulsions or suspensions, includingsaline and buffered media. Parenteral vehicles can include sodiumchloride solution, Ringer's dextrose, dextrose and sodium chloride,lactated Ringer's or fixed oils. Intravenous vehicles can includevarious additives, preservatives, or fluid, nutrient or electrolytereplenishers (See, generally, Remington's Pharmaceutical Science, 16thEdition, Mack, Ed. 1980). For inhalation, the compound is solubilizedand loaded into a suitable dispenser for administration (e.g., anatomizer, nebulizer or pressurized aerosol dispenser).

Example 1

Methods

Laser photocoagulation (532 nm, 200 mW, 100 ms, 75 μm) (OCULIGHT™ GL,Index Corporation) was performed (volume studies: 3/eye; proteinanalyses/flow cytometry: 12/eye) on both eyes of each animal to induceCNV (choroidal neovascularization). CNV volumes were measured byscanning laser confocal microscope (TCS SP, Leica) with 0.5%FITC-Griffonia simplicifolia Isolectin B4 (Vector Laboratories). CNV wasinduced by laser injury in C57BL/6J and Ccr3^(−/−) mice and volumesmeasured 7 days later by confocal evaluation of Griffonia simplicifoliaIsolectin B4 staining of RPE-choroid flatmounts. Neutralizing antibodies(Ab) against CCR3, eotaxin (CCL-11), eotaxin-2 (CCL-24), RANTES, MCP-3or control goat IgG or rat IgG_(2a) were injected into the vitreoushumor following injury.

Flow cytometry was used to determine the numbers of eosinophils, mastcells and macrophages in the choroid, expression of CCR3 by various celltypes in the eye, and the cell cycle state of choroidal endothelialcells (CECs) in vivo. Suspensions of cells isolated from mouseRPE/choroid by incubation with collagenase D (20 U/ml; RocheDiagnostics) treatment were incubated in Fc block (0.5 mg/ml; BDPharmingen) for 15 min. on ice. Rat antibody to mouse CCR3 (1:250; SantaCruz) coupled with PE-donkey antibody to rat IgG (1:250; JacksonImmunoresearch) were used to quantify cell surface receptor expressionon choroidal endothelial cells, defined by CD31⁺ VEGFR-2⁺ expression,gated by FITC-conjugated rat antibody to mouse CD31 (1:250; BDBiosciences) and PerCP-Cy5.5-conjugated rat antibody to mouse CD11b(1:50; BDBiosciences). Macrophages, neutrophils, eosinophils and mastcells were defined as F4/80⁺CD11c⁻, Gr-1⁺F4/80⁻, CCR3^(hi)CD3⁻CD1171^(int)CD49d⁺ and CCR31^(int)CD3⁻ CD117^(hi)CD49d⁺ cells,respectively. DNA content for cell cycle was analyzed after incubationwith propidium iodide (0.05 mg/ml; Molecular Probes) containing 0.1%TRITON™ X-100 and RNase A (0.1 mg/ml; Roche).

Because the probability of each laser lesion developing CNV isinfluenced by the group to which it belongs, the mouse, the eye, and thelaser spot, the mean lesion volumes were compared using a linear mixedmodel with a split plot repeated measures design. The whole plot factorwas the genetic group to which the animal belonged while the split plotfactor was the eye. Statistical significance was determined at the 0.05level. Post hoc comparison of means was constructed with a Bonferroniadjustment for multiple comparisons.

Results

As illustrated by FIG. 1, the number of CCR3 receptors on choroidalendothelial cells in vivo following laser injury (red) is significantlygreater than the number before injury (green), indicating upregulationof CCR3 receptors on these cells. Eosinophils or mast cells are theprincipal cells in most systems that respond to CCR3. However, thenumber of eosinophils and mast cells in the choroid was unaffected byinjury or CCR3 Ab (FIG. 4). CCR3 Ab did not inhibit choroidal macrophageinfiltration following injury (FIG. 5), indicating that laser injury isnot working by anti-inflammatory means. As illustrated by FIG. 3, CCR3Ab suppressed CNV volume in C57BL/6J mice by nearly 60% in adose-dependent and statistically significant manner compared to vehiclecontrol (PBS) and control antibody (rat IgG2a). FIG. 2 demonstrates thatCCR3 Ab blockade, but not control antibody (rat IgG2a), inhibitedproliferation (S phase) of CECs (choroidal endothelial cells) in vivofollowing laser injury. Experiments in Ccr3^(−/−) mice confirmed theseresults. Of the CCR3 ligands, blockade of only eotaxin (45%) oreotaxin-2 (70%) suppressed CNV in C57BL/6J mice compared to controlantibodies (all Ps<0.001). Experiments in Ccl11^(−/−) and Ccl24^(−/−)mice confirmed these results.

These findings demonstrate that CCR3 receptor promotes angiogenesis notvia leukocyte modulation but rather by direct effects on CECs. Thus,CCL-11, CCL-24, and CCR3 are new targets for neovascular AMD(age-related macular degeneration).

All references cited in this disclosure are incorporated by reference tothe same extent as if each reference had been incorporated by referencein its entirety individually.

While the invention has been described in detail and with reference tospecific embodiments thereof, it will be apparent to one skilled in theart that various variations and modifications can be made thereinwithout departing from the sprit and scope thereof. All such variationsand modifications are intended to be included within the scope of thisdisclosure and the present invention and protected by the followingclaims.

I claim:
 1. A method of treating ocular angiogenesis in a mammal in needof treatment for ocular angiogenesis comprising exposing a choroidalcell of the mammal to a CCR3-inhibitory effective amount of an organicmolecule that inhibits the activity of CCR3, wherein ocular angiogenesisis treated in the mammal.
 2. The method of claim 1, wherein thechoroidal cell is a choroidal endothelial cell.
 3. The method of claim1, wherein the organic molecule is orally administered to the mammal. 4.The method of claim 1, wherein the organic molecule is intravenouslyadministered to the mammal.
 5. The method of claim 1, wherein theorganic molecule is intraocularly injected into the mammal.
 6. Themethod of claim 1, wherein the organic molecule has a molecular weightless than
 1000. 7. The method of claim 1, wherein the organic moleculehas a molecular weight less than
 500. 8. The method of claim 3, whereinthe mammal is a human.
 9. The method of claim 4, wherein the mammal is ahuman.
 10. The method of claim 5, wherein the mammal is a human.
 11. Themethod of claim 1, wherein the mammal in need of treatment for ocularangiogenesis has age-related macular degeneration.
 12. The method ofclaim 1, wherein the mammal in need of treatment for ocular angiogenesishas choroidal neovascularization.